Vibrio species are predominantly aquatic bacteria. While many of them are non-pathogenic, some vibrios are associated with severe disease in humans. Vibrio cholerae, in particular, has a long history of causing epidemic and pandemic cholera. Cholera results from colonization of the small intestine by V. cholerae, where it produces a potent toxin that results in massive water loss and electrolyte imbalance. In nature, V. cholerae may be found free-living, but its survival appears to be favored by specific associations with other organisms. Some of the genes required for V. cholerae to form these associations and persist in its aquatic ecosystem are also involved in promoting disease in humans. In vitro studies have suggested that protozoans may both use V. cholerae as a food source and promote V. cholerae survival, but the nature of these interactions is not well understood. Our studies will focus on the relationship between V. cholerae and the model protozoan amoeba Dictyostelium discoideum, and our laboratories have combined expertise in these two organisms. Our hypothesis is that V. cholerae is subject to predation by amoebae in the environment and that production of virulence factors such as cholera toxin helps protect V. cholerae from predation and enhances its survival. Further, we propose that D. discoideum can be used as a model for interaction of V. cholerae with amoebae. We will use genetic, biochemical, and cellular biology approaches to dissect the relationship between D. discoideum and V. cholerae. Analysis of mutants and genetic screens will be used to identify the V. cholerae genes required for survival and persistence in the presence of D. discoideum, and we will use cellular biology techniques to begin to elucidate the interactions between D. discoideum and V. cholerae. The specific aims are to (i) identify V. cholerae genes involved in resistance to predation by amoebae, (ii) determine the mechanism of survival of V. cholerae within D. discoideum, and (iii) determine whether V. cholerae grown in the presence of the amoeba are infectious or hypervirulent. Understanding this predator/prey relationship will not only provide basic information about V. cholerae in the environment, but also may provide clues to methods for better controlling the environmental sources of V. cholerae and thereby help prevent epidemics of cholera. PUBLIC HEALTH RELEVANCE: Vibrio cholerae is a major cause of morbidity and mortality worldwide. This bacterium has a reservoir in nature and periodically re-emerges to cause epidemics and pandemics of cholera. In order to persist in the wild, V. cholerae must be able to resist predation by amoebae. The proposed studies to determine the mechanisms of resistance to predation will lead to a better understanding of the ecology of V. cholerae. This may allow the development of strategies to control the source of epidemic V. cholerae.